Safe Staphylococcal Platform for the Development of Multivalent

Safe Staphylococcal Platform for the Development of Multivalent Nanoscale Vesicles against Viral Infections. Jizhen Yuan†, Jie ... Publication Date ...
0 downloads 13 Views 3MB Size
Subscriber access provided by UNIV OF MISSOURI ST LOUIS

Communication

Safe staphylococcal platform for the development of multivalent nanoscale vesicles against viral infections yuan jizhen, Jie Yang, Zhen Hu, Yi Yang, Weilong Shang, Qiwen Hu, Ying Zheng, Huagang Peng, Xiaopeng Zhang, Xinyu Cai, Junmin Zhu, Ming Li, Xiaomei Hu, Renjie Zhou, and Xiancai Rao Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b03893 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 18, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Nano Letters is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

1

Safe staphylococcal platform for the development of multivalent nanoscale

2

vesicles against viral infections

3 4 5

Jizhen Yuan#, Jie Yang#, Zhen Hu#, Yi Yang#, Weilong Shang#, Qiwen Hu#, Ying

6

Zheng#, Huagang Peng#, Xiaopeng Zhang#, Xinyu Cai⊥, Junmin Zhu#, Ming Li#,

7

Xiaomei Hu#, Renjie Zhou⊥,*, Xiancai Rao#,*

8 9 10

#

Department of Microbiology, College of Basic Medical Sciences, Third Military

11

Medical University, 30# Gaotanyan St., Shapingba District, Chongqing 400038, P. R.

12

China.

13 14



Department of Emergency, Xinqiao Hospital, Third Military Medical University, 83# Xinqiao St., Shapingba District, Chongqing 400037, P. R. China.

15 16 17 18 19 20 21 22

Running title: Platform for the development of nanoscale vesicles

23 24 25 26 27 28 29 30 1

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 21

31

ABSTRACT

32

Many viruses often have closely related yet antigenically distinct serotypes. An ideal

33

vaccine against viral infections should induce a multivalent and protective immune

34

response against all serotypes. Inspired by bacterial membrane vesicles (MVs) that

35

carry different protein components, we constructed an agr locus deletion mutant of

36

the Staphylococcus aureus strain (RN4220-∆agr) to reduce potential toxicity.

37

Nanoscale vesicles derived from this strain (∆agrMVs) carry at least four major

38

components that can deliver heterologous antigens. These components were each

39

fused with a triple FLAG tag, and the tagged proteins could be incorporated into the

40

∆agr

41

and (2.89 ± 0.74)% of the total

42

PdhA-FLAG, and Eno-FLAG, respectively. With two DENV envelope E domain III

43

proteins (EDIIIconA and EDIIIconB) as models, the DENV EDIIIconA and

44

EDIIIconB delivered by two staphylococcal components were stably embedded in the

45

∆agr

46

against all four DENV serotypes. Sera from immunized mice protected Vero cells and

47

suckling mice from a lethal challenge of DENV-2. This study will open up new

48

insights into the preparation of multivalent nano-sized viral vaccines against viral

49

infections.

MVs. The presentation levels were (3.43 ± 0.73)%, (5.07 ± 0.82)%, (2.64 ± 0.61)%, ∆agr

MV proteins for Mntc-FLAG, PdhB-FLAG,

MVs. Administration of such engineered

∆agr

MVs in mice induced antibodies

50 51 52 53 54

KEYWORDS: Staphylococcus aureus; membrane vesicle; agr; vaccine; dengue virus

55 56 57 58 59 60 2

ACS Paragon Plus Environment

Page 3 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

61

Vaccination is the most effective strategy for preventing viral diseases. However,

62

many viruses often have closely related yet antigenically distinct serotypes or

63

genotypes, such as poliovirus,1 herpes simplex virus,2 human papillomavirus,3 and

64

dengue virus (DENV).4 Developing vaccines for diseases caused by such

65

polyserotypic viruses is challenging.5 In general, the viral vaccine for each serotype or

66

genotype is prepared and subsequently administrated separately or in a combined

67

formulation. The development of a versatile platform for the generation of multivalent

68

viral vaccines is high priority.

69

Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria have been

70

studied for nearly five decades and have since emerged as attractive and effective

71

vaccines or delivery systems.6–9 Nano-sized OMVs can be gradually released from the

72

outer membrane of Gram-negative bacteria cell walls. OMVs are also secreted from

73

pathogens, such as Burkholderia pseudomallei,7 Shigella boydii,8 Salmonella enterica

74

serovar Typhimurium,9 and Neisseria meningitides.10 These OMVs induce potent

75

protective immune responses against certain pathogens. Moreover, OMVs from

76

Gram-negative bacteria have intrinsic adjuvant roles.8,11 Several recombinant

77

Meningococcus B antigens formulated with homologous strain OMVs increase

78

immunogenicity.10 The serine protease HtrA of Chlamydia muridarum expressed with

79

a pET21b+ vector is accumulated in the OMVs of Escherichia coli, and the resulting

80

OMVs can induce neutralizing antibodies in an in vitro infectious assay, whereas the

81

purified recombinant HtrA cannot.12 However, OMV vaccination is limited by the

82

incorporation of lipopolysaccharide (LPS) or lipooligosaccharide (LOS) into the

83

bilayer of OMVs.13 Due to the lack of outer membrane and the existence of rigid cell

84

walls, direct evidence on membrane vesicle (MV) formation in Gram-positive bacteria,

85

such as Staphylococcus aureus and Bacillus subtilis, was only recently demonstrated

86

through transmission electron microscopic and proteomic analyses.14,15 MVs of

87

Gram-positive bacteria share common features with the OMVs of Gram-negative ones;

88

however, the mechanisms by which Gram-positive bacteria secrete MVs are largely

89

unknown.14 Both MVs and OMVs are spherical non-replicating nanoparticles with the

90

diameter of 20–200 nm and contain phospholipid bilayers that are incorporated with 3

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 21

91

various bacterial proteins and lumens carrying periplasmic constituents.14,16,17

92

Mycobacterium tuberculosis H37Rv MVs not only elicited protection as well as live

93

BCG vaccine but also boosted BCG vaccine efficacy.18 When intraperitoneally

94

injected, Streptococcus pneumoniae MVs protected mice from the homologous strain

95

and other pathogenic serotype of S. pneumoniae infections.19 However, application

96

studies on nanoscale MVs derived from Gram-positive bacteria to protect

97

heterologous infections are not available.17

98

We proposed a safe staphylococcal platform in which the toxicity of MVs was

99

attenuated (∆agrMVs), and several ∆agrMV-incorporated components were characterized

100

as capable of delivering heterologous viral antigens that resulted in the release of

101

multivalent nanoscale ∆agrMVs against viral infections (Scheme 1). Using two DENV

102

consensus peptides as model antigens, we demonstrated that the successful

103

incorporation of DENV peptides into the staphylococcal

104

administration of these engineered

105

immune response against all the serotypes of DENV. Our data revealed the suitability

106

of the staphylococcal

107

MV vaccines and illustrated the potential of using this platform in developing

108

multivalent viral vaccines.

∆agr

∆agr

∆agr

MVs and the

MVs induced a multivalent and protective

MV platform for developing a new generation of nanoscale

109 110 111

Scheme 1. Schematic representation of the multivalent nanoscale strategy. 4

ACS Paragon Plus Environment

∆agr

MV generation

Page 5 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

112

Vesicle release is a ubiquitous process that occurs during normal bacterial growth

113

and has been extensively characterized in Gram-negative bacteria (OMVs).20 The

114

secretion of MVs from S. aureus was reported for the first time in 2009,14 and such

115

report not only broke the assumption that Gram-positive bacteria could hardly release

116

MVs because of the existence of thick and rigid cell walls but also provided an

117

alternative to OMVs for developing potential vaccines for clinical applications.

118

Taking advantage of the available genome sequences, MVs of several laboratory S.

119

aureus strains (Table S1) were separately isolated, as previously described.21

120

Transmission electron microscopy (TEM) revealed that all the tested S. aureus strains

121

can secrete nano-sized MVs with the diameter of 49.8 ± 17.4 nm (Figures 1A and S1).

122

Although the amount of vesicle released between strains has no significant difference

123

(Figure 1B), the protein components that are incorporated into the staphylococcal

124

MVs are strain-dependent (Figures 1C and 1D). Among the tested strains, RN4220

125

has a frame shift at the 3′-terminal of the agrA gene that could delay the

126

agr-controlled expression of virulence factors and may produce safe MVs.22 In

127

addition, RN4220 has defects in type I restriction-modification system and is more

128

convenient for performing genetic operations.23 Therefore, S. aureus RN4220 was

129

selected as the starting strain in this study.

130 5

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 21

131

Figure 1. Analysis of MVs derived from different S. aureus strains. (A) The MVs produced by

132

S. aureus strains observed under the transmission electron microscope. The bars representing

133

200 nm were indicated. (B) The production of MVs from each S. aureus strain was shown as total

134

proteins (µg) in MVs derived from 1 L overnight cultures determined by the Bradford assay; n = 3

135

experiments. (C) SDS-PAGE analysis of MVs derived from different S. aureus strains. The

136

molecular weights of a protein marker were indicated on the left. (D) The abundance of proteins in

137

the MVs of S. aureus was analyzed by the plot lane tool of ImageJ software version 1.46 (National

138

Institutes of Health, USA).

139 140

The production of S. aureus strain RN4220 MVs achieved its peak at 22 h

141

post-culture (Figure 2A). Although lacking in LPS and LOS, the MVs derived from S.

142

aureus could still contain some species-specific virulence factors that affect the safety

143

of a potential vaccine.24 Dose-dependent experiments revealed that the survival rate

144

was 0%, 10%, 20%, 50%, 80%, and 100% after BALB/c mice were intraperitoneally

145

challenged with 80, 64, 51, 41, 33, and 26 µg of the wild-type RN4220-derived MVs

146

(wtMVs), respectively (Figure 2B). According to the Bliss method, the calculated LD50

147

for

148

intraperitoneal administration. The high toxicity of

149

fact that around 100 bacterial proteins are incorporated into S. aureus wtMVs,24, 25 and

150

several toxic components are enriched during the MV concentration procedure.26 For

151

the safety of MVs used as nano-sized vaccine delivery vehicles, characterizing every

152

toxic component in wtMVs and eliminating its effect is a great challenge. Our strategy

153

was to delete the whole locus of staphylococcal agr (Figure S2), a key quorum

154

sensing system consisting of agrB, agrD, agrC, agrA, and RNAIII in S. aureus,27 to

155

reduce the expression of virulence factors. As expected, the mice survival rate was

156

100% even after they were intraperitoneally injected with 200 µg of the agr-deleted

157

RN4220-derived MVs (∆agrMVs) (Figure 2B). We then examined the systemic

158

inflammatory indexes IL-6 and TNFα to assess the potential toxicities of

159

∆agr

160

reduced after intraperitoneal injection of same dose of ∆agrMVs (Figure 2C, left panel).

wt

MVs was 2.1 mg/kg with a 95% confidence interval of 1.85–2.35 mg/kg for

MVs in vivo. Compared with

wt

wt

MVs can be explained by the

wt

MVs and

MVs, the levels of serum IL-6 were significantly 6

ACS Paragon Plus Environment

Page 7 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

161

However, the significantly increased TNFα level was only observed in the high-dose

162

wt

163

∆agr

164

mouse challenged with 40 µg

165

aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase

166

(LDH) were also determined to further evaluate the liver toxicities of

167

∆agr

168

levels of ALT, AST, and LDH, whereas 20 µg

169

levels compared with 100 µg

170

RN4220-∆agr (∆agrMVs) exhibit attenuation in the mouse model and are safe for

171

antigen delivery vehicles.

MV-challenged group (Figure 2C, right panel). Even challenged with a high dose of MVs (100 µg), mouse serum IL-6 and TNFα levels were still lower than those of wt

MVs (Figure S3). In addition, serum alanine

MVs. As shown in Figure 2D, both 20 µg and 100 µg

∆agr

wt

∆agr

wt

MVs and

MVs could not alter the

MVs significantly increased their

MVs. Taken together, MVs derived from S. aureus

172 173

Figure 2. Deletion of agr locus attenuated the MVs of S. aureus. (A) Production of S. aureus

174

RN4220

175

BALB/c mice (6–8 weeks old) challenged with wtMVs (80, 64, 51, 41, 33, and 26 µg) and ∆agrMVs

176

(200 µg) (n = 10 for each group). (C) Mouse serum levels of IL-6 and TNFα 6 h

177

post-intraperitoneal injection of wtMVs (10, 20, and 40 µg) and ∆agrMVs (10, 20, and 40 µg) (n = 4

wt

MVs during different growth stages (n = 3 for each time point). (B) Survival rates of

7

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 21

wt

178

for each dose). (D) Hepatic injury indexes after intraperitoneal injection of

MVs (20 µg) and

179

∆agr

180

group). Data are presented as the mean ± SD. n.s. represented no significance, * P < 0.05, **

181

P < 0.01, and *** P < 0.001.

MVs (20 µg, 100 µg). CCl4 (10% in corn oil) was used as a positive control (n = 4 for each

182

SDS-PAGE revealed that the protein patterns of RN4220-∆agr and wild-type

183

∆agr

184

RN4220 were very similar; however, the protein components of

185

some extent compared with those of the

186

performed a proteomic analysis to explain why the agr locus deletion reduced the

187

toxicity of MVs. A total of 92 proteins were identified in the

188

components were characterized in the ∆agrMVs (Table S2). wtMVs and ∆agrMVs had 61

189

common proteins (Figure 3B). This phenomenon has also been observed in a recent

190

study where the MVs from three S. aureus isolates carried 25 common proteins and

191

60 strain-specific proteins.28 Proteomics analysis also revealed that the most abundant

192

protein in

193

modified protein abundance index (emPAI) value of 93876.4, followed by PSMα

194

(2259 Da, emPAI = 6.9) and alpha-hemolysin (emPAI = 1.02) (Figure 3C). However,

195

these virulence factors were absent in the

196

Table S2), thereby supporting the survival rate of the ∆agrMV-challenged mouse model

197

(Figure 2B). Gene Ontology (GO) analysis revealed that the major components in

198

∆agr

199

metabolic process with catalytic activity (Figure S4). Moreover, Keiser et al.29

200

reported that detoxifying the LOS of OMVs by disabling the lpxL2 gene in group B

201

Meningococcus results in reduced viability of the bacteria; however, deletion of agr

202

locus in S. aureus (RN4220-∆agr) would not affect bacterial growth (Figure S5A),

203

and

204

RN4220-∆agr (Figure S5B), thereby indicating a successfully engineered host of S.

205

aureus RN4220-∆agr for the production of nanoscale vesicles.

wt

MVs varied to

MVs (Figure 3A). We subsequently

wt

MVs, and 119

wt

MVs was delta-hemolysin (5009 Da), which had an exponentially

∆agr

MVs from RN4220-∆agr (Figure 3D,

MVs were cytosolic and cytoplasmic proteins that are involved in the cellular

∆agr

MV protein patterns were similar after logarithmic growth of the engineered

8

ACS Paragon Plus Environment

Page 9 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

206 207

Figure 3. Proteomic analysis of proteins incorporated in wtMVs and ∆agrMVs. (A) Comparison

208

of protein profiles between S. aureus RN4220 and RN4220-∆agr, as well as their derived MVs

209

(wtMVs and ∆agrMVs). The sizes of a protein marker were indicated on the left. The major bands of

210

∆agr

211

indicated by a black arrow. (B) Proteins detected in the proteomic analysis with liquid

212

chromatography-tandem mass spectrometry (LC-MS/MS) in the MVs derived from S. aureus

213

RN4220 and RN4220-∆agr. The number (percent) of the identical and different proteins in the

214

wt

215

protein in wtMVs was delta-hemolysin with an emPAI value of 93876.4, followed by PSMα and

216

alpha-hemolysin, as indicated. (D) Major components detected in

217

The major components in ∆agrMVs seemed scattered with the most predominant protein of enolase

218

(47,177 Da, emPAI = 3.4).

MVs were indicated by black triangles, and the abundant band that was absent in ∆agrMVs was

MVs and ∆agrMVs was indicated. (C) Major components detected in wtMVs. The most abundant

∆agr

MVs with the emPAI > 1.

219 220

The major components that corresponded to the rich bands in SDS-PAGE gel were

221

shared by wtMVs and ∆agrMVs (Figure 3A). Eight abundant bands with variable sizes

222

in the

223

by LC-MS/MS to select the optimal components that can deliver heterologous

224

antigens. Seven of these proteins were also identified in the proteomic analysis of

∆agr

MV gel (Figure 3A, indicated by black triangles) were further characterized

9

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

225

wt

226

potential capacity of these proteins in delivering heterologous antigens, a DNA

227

sequence encoding 3×FLAG tag (DYKDHDGDYKDHDIDYKDDDDK) was

228

chemically synthesized and genetically in-frame fused with the 3′-terminal of each

229

protein encoding gene of S. aureus RN4220-∆agr through homologous recombination.

230

Four

231

RN4220-∆agr/Mntc-FLAG, RN4220-∆agr/PdhB-FLAG, RN4220-∆agr/PdhA-FLAG,

232

and RN4220-∆agr/Eno-FLAG (Figure S6). These fusion proteins could be detected in

233

the knocked-in strains and incorporated into the ∆agrMVs by using mAb against FLAG

234

(Figures 4A and 4B). Quantitative Western blot showed that the FLAG-fused proteins

235

represented (3.43 ± 0.73)%, (5.07 ± 0.82)%, (2.64 ± 0.61)%, and (2.89 ± 0.74)% of

236

the total

237

Eno-FLAG, respectively (Figures 4C and S7). However, the reasons for the failure of

238

RplE (20.3 kDa), PdhC (46.3 kDa), and GlnA (50.9 kDa) in carrying heterologous

239

antigens remained unclear. The failure might either be due to the technical problems

240

or the gene itself for S. aureus survival. At least four abundant components with

241

variable sizes in

242

∆agr

243

attenuating toxicity while possessing a heterologous antigen delivery capability.

MVs and

∆agr

Page 10 of 21

MVs, and their emPAI values were above 1.0 (Table S3). To test the

knocked-in

strains

were

successfully

constructed,

designated

as

∆agr

MV proteins for Mntc-FLAG, PdhB-FLAG, PdhA-FLAG, and

∆agr

MVs are capable of delivering heterologous antigens into the

MVs, thereby highlighting the staphylococcal platform as an effective mode of

244 10

ACS Paragon Plus Environment

Page 11 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

245

Figure 4. Characterization of protein components’ potential for delivery of heterologous

246

antigens. (A) Western blot analysis of 3×FLAG-tagged proteins in the total cell lysates using

247

anti-FLAG monoclonal antibodies. The full-length blots are presented in Figure S10. (B) Western

248

blot analysis of 3×FLAG-tagged proteins in total

249

Experimental section. (C) Abundance of FLAG-tagged protein in the

250

of

251

and analyzed by the plot lane tool of the ImageJ software version 1.46 (right panel). Certain

252

FLAG-tagged proteins were detected by Western blot (central panel); their amounts were

253

determined using a standard curve from the Western blot for pure Eno-FLAG in

254

S7) and indicated on the top as a percentage to the total

255

corresponding to the band in gel was indicated by a black triangle.

∆agr

MVs prepared as described in the ∆agr

MVs. The total proteins

∆agr

MVs derived from each knocked-in strain were separated by 12% SDS-PAGE (left panel)

∆agr

MVs (Figure

∆agr

MV proteins. The relative peak

256

To test the validity of staphylococcal platform for the generation of multivalent

257 258

∆agr

259

DENV serotypes were selected as model antigens. DENV has four serotypes, which

260

were designated as DENV-1, -2, -3, and -4. These serotypes are widely distributed in

261

tropical and subtropical regions that affect approximately 3.6 billion people

262

worldwide.30 Previous reports indicated that EDIII is the major protective domain for

263

prevention of DENV infections.31,32 Among the four DENV serotypes, the EDIIIs of

264

DENV-1 and -3 are the most similar to each another, whereas DENV-2 is the closest

265

to DENV-4.33 To simplify the construction process, physicochemical property method

266

(PCP method)33,34 was used to generate two PCP-consensus EDIII sequences

267

representing DENV-1/-3 (EDIIIconA) and DENV-2/-4 (EDIIIconB). Four EDIII

268

PCP-consensus sequences (DENV-1–4con, Figure S8A) were first created based on

269

the individual alignments of 356 DENV-1, 147 DENV-2, 146 DENV-3, and 181

270

DENV-4 sequences derived from GenBank (http://www.ncbi.nlm.nih.gov). Then,

271

EDIIIconA was obtained by PCP method using DENV-1con and DENV-3con as

272

inputs, analogously to obtain EDIIIconB that represents the DENV-2/-4 (Figure S8B).

273

The coding sequences of EDIIIconA and EDIIIconB were deduced according to

274

the codon usage bias of S. aureus RN4220 (Figure S8C) and were chemically

MVs, the envelope E protein domain III (EDIII, residues 296–395) of different

11

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

275

synthesized to decrease the effect of codon bias on the expression of target fusion

276

protein. Then, EDIIIconA and EDIIIconB sequences were in-frame fused with the

277

3′-terminal of eno and pdhB genes in the genomic DNA of S. aureus RN4220-∆agr,

278

respectively (Figure 5A). The expected productions of Eno-EDIIIconA and

279

PdhB-EDIIIconB fusion proteins were confirmed by Western blot analysis in both

280

total cell lysates of the engineered bacteria and the total ∆agrMVs (Figures 5B and 5C).

281

Immunoelectron microscopy (IEM) revealed that DENV EDIII antigens showed

282

efficient labeling using anti-EDIII and were mainly carried in the lumen or presented

283

on the surface of

284

can be delivered into the

285

vesicular components of S. aureus RN4220-∆agr.

∆agr

MVs (Figures 5D and 5E). The viral antigen (dengue EDIIIcon) ∆agr

MVs through the orientation of characterized major

286

12

ACS Paragon Plus Environment

Page 12 of 21

Page 13 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

287 288

Figure 5. Expression and identification of the Eno-EDIIIconA and PdhB-EDIIIconB fusion

289

proteins. (A) Schematic representation of the size changes when Eno and PdhB fused with

290

EDIIIconA and EDIIIcinB, respectively. (B) Western blot analysis of target fusion proteins in the

291

total cell lysates of RN4220-∆agr and RN4220-∆agr-EDIIIconA/B using anti-Eno antibodies (left

292

panel), anti-PdhB antibodies (central panel), and anti-DENV-2con antibodies (right panel). The

293

full-length blots are presented in Figure S10. (C) Western blot analysis of fusion proteins in the

294

total

295

panel), anti-PdhB antibodies (central panel), and anti-DENV-2con antibodies (right panel). (D)

296

Immunoelectron micrographs of EDIIIconA/B contained

297

Experimental section. Micrographs show efficient labeling of EDIIIconA/B-fused proteins with

298

mouse-anti-DENV-2con antibodies (indicated by black arrows). (E) Immunoelectron micrographs

299

of ∆agrMVs derived from RN4220-∆agr served as negative control.

∆agr

MVs of RN4220-∆agr and RN4220-∆agr-EDIIIconA/B using anti-Eno antibodies (left

∆agr

MVs detected, as described in the

300 301

Given the potent component-mediated delivery of viral antigens into the ∆agrMVs,

302

we speculated that engineered dengue ∆agrMVs can evoke host immune responses. The

303

EDIIIconA/B contained multivalent ∆agrMV-induced higher titers of antibodies against

304

the recombinant EDIII (rEDIII) proteins of all four DENV serotypes than the

305

EDIIIconB contained

306

multivalent

∆agr

MVs

∆agr

MVs did (Figure 6). With only prime vaccination, the

induced

higher

binding

antibodies

13

ACS Paragon Plus Environment

than

the

rEDIII

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 21

∆agr

307

protein-challenged (Figure 6A). EDIIIconB contained

308

antibodies against the rEDIII of DENV-2 and DENV-4. Similar results were observed

309

in the prime-boost vaccination schedule (Figures 6B and 6C), which were concordant

310

with the fact that EDIIIconB (representing DENV-2/-4) contains less epitopes against

311

DENV-1 and DENV-3. In the three prime-boost schedules, EDIIIconA/B contained

312

multivalent

313

serotypes of DENV regardless of the use of adjuvants (Figures 6B and 6C), thereby

314

suggesting that staphylococcal ∆agrMVs may have an adjuvant effect similar to that of

315

the OMVs derived from Gram-negative bacteria.8,11 The engineered ∆agrMVs carrying

316

dengue EDIII antigens can effectively evoke humoral immune responses in mice with

317

intrinsic adjuvant activity. However, the mechanism by which staphylococcal ∆agrMVs

318

display adjuvant capacity requires further investigation.

∆agr

MV-evoked higher binding

MV-induced similar humoral immune responses against all the four

319 320

Figure 6. Capture ELISA detected the binding antibodies in mouse sera against recombinant

321

EDIII (rEDIII) antigens (DENV-1–4con). (A) Sera derived from immunized mice with a single

322

vaccination of EDIIIconA/B or EDIIIconB ∆agrMVs without adjuvants. Sera from mice challenged

323

with DENV-1–4con (rEDIII) and PBS served as positive and negative controls, respectively. (B) 14

ACS Paragon Plus Environment

Page 15 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

324

Sera derived from immunized mice with thrice prime-boost schedule of EDIIIconA/B or

325

EDIIIconB

326

prime-boost schedule of EDIIIconA/B or EDIIIconB

327

arbitrary units. Data represent three independent experiments and expressed as mean ± SD.

∆agr

MVs without adjuvants. (C) Sera derived from immunized mice with thrice ∆agr

MVs with Freund’s adjuvants. AU,

328 329

To test whether the immunized mouse sera could neutralize virus infection, plaque

330

reduction neutralization test (PRNT)35 was performed. The neutralizing effect of

331

EDIIIconA/B contained

332

dose-dependent. The 320 times diluted immune sera could still inhibit approximately

333

75% formation of DENV-2 plaques, whereas the control sera at 1:40 dilution could

334

only offer an inhibition of less than 20% (Figure 7A). Immunofluorescence assay

335

(IFA) was also used to monitor the multiplication of DENV-2 in Vero cells after

336

neutralizing the virus with immunized murine sera against EDIIIconA/B multivalent

337

∆agr

338

DENV-2 infection, whereas the normal BALB/c mouse sera could not (Figure 7B).

339

These results were consistent with those of PRNT (Figure 7A). Furthermore, the

340

protective activity of the immunized sera was also evaluated in vivo. Approximately

341

80% of the tested suckling mice were protected from the lethal dose challenge of

342

DENV-2, which was pre-incubated with 1:80 diluted immunized murine sera. All the

343

control mice died 10 days after they were post-challenged by normal sera pretreated

344

virus (Figure 7C). EDIIIconA/B-loaded ∆agrMVs can induce dengue-specific humoral

345

immune responses and confer effective protection against DENV-2 infection.

∆agr

MV-immunized mouse sera (three prime-boost) was

MVs. The immunized sera diluted at 1:80 could completely protect Vero cells from

15

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 21

346 ∆agr

347

Figure 7. Protective roles of EDIIIconA/B multivalent

MV sera against DENV-2. (A)

348

Neutralizing activities of immunized sera against DENV-2 determined by plaque reduction

349

neutralization test (PRNT). Diluted sera from immunized mice treated thrice with prime-boost of

350

EDIIIconA/B

351

cells. Results were expressed as the percentage blocking (PB%) in PRNT. PB% was calculated as

352

described in the Experimental section. Normal mouse sera served as negative control. (B)

353

Multiplication of DENV-2 in Vero cells tested with an immunofluorescence assay (IFA). The

354

dilutions of sera from the EDIIIconA/B

355

were used as negative control. (C) Survival rates of BALB/c suckling mice challenged with

356

DENV-2 pre-incubated with 1:80 diluted sera from EDIIIconA/B

357

circle) or with sera from normal mice (filled square).

∆agr

MVs without adjuvants were incubated with DENV-2 before infection to Vero

∆agr

MV-immunized mice were indicated, and normal sera

∆agr

MV-immunized mice (filled

358 359

In conclusion, the applicability of the major components of S. aureus MVs for

360

delivering serotypic viral antigens as multivalent nanoscale vaccines was tested. We

361

proposed an agr-deleted S. aureus strain with four well-characterized MV components

362

for the delivery of heterologous antigens to produce multivalent vaccines. This safe 16

ACS Paragon Plus Environment

Page 17 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

363

platform has a broad range of biotechnological applications, including the generation

364

of multivalent vaccines against viruses with several serotypes and/or different viruses

365

(Figure S9). However, this platform has some limitations. It may not be particularly

366

useful for expressing glycosylated antigens. The protective viral peptides without

367

glycosylation, such as DENV EDIIIs, are satisfied. In addition, the production of

368

∆agr

369

a reasonable scale for pharmaceutical applications, and further biotechnological

370

optimization is needed. Our study provides an innovative attempt to use

371

Gram-positive bacteria-released nanoscale MVs as immunologic tools for fighting

372

viral infections.

MVs using engineered RN4220-∆agr may rarely be sufficiently efficient to achieve

373 374 375 376

ASSOCIATED CONTENT

377

Supporting Information

378

Detailed description of the Experimental section and additional data are provided.

379

Figures S1–S10, Tables S1–S5.

380 381

AUTHOR INFORMATION

382

Corresponding Authors

383

*X. R. ([email protected]); R. Z. ([email protected]).

384 385

Author Contributions

386

X.R., R.Z., and X.H. conceived and designed the experiments. J.Y., Z.H., W.S., Y.Z.,

387

H.P., X.C., and X.Z. performed the experiments. J.Y., X.R., Q.H., M.L., J.Z., and Y.Y.

388

analyzed the data. J.Y., Y.Y., and X.R. wrote the manuscript. All authors discussed the

389

results and commented on the manuscript. The principal investigator is X.R. and R.Z.

390 391

ORCID

392

Jizhen Yuan, 0000-0003-4693-9534; Jie Yang, 0000-0002-2886-9774; Zhen Hu, 17

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Yang,

0000-0002-3707-5052;

Page 18 of 21

393

0000-0002-1077-6991;

Yi

394

0000-0002-0752-5016;

Qiwen

395

0000-0002-9481-2733; Huagang Peng, 0000-0002-3482-0213; Xiaopeng Zhang,

396

0000-0002-3880-1810;

Xinyu

Cai,

397

0000-0002-1652-5421;

Ming

Li,

398

0000-0003-2831-1241;

Renjie

Zhou,

399

0000-0002-9905-760X.

Hu,

Weilong

Shang,

Ying

Zheng,

0000-0001-6689-6194;

0000-0002-3045-6899; 0000-0003-1662-6465; 0000-0003-3046-567X;

Junmin

Zhu,

Xiaomei

Hu,

Xiancai

Rao,

400 401

Notes

402

The authors declare no competing financial interests.

403 404

ACKNOWLEDGMENTS

405

We thank Prof. Daoguo Zhou (Purdue University) for critical reading of the

406

manuscript. This work was supported by the national natural science foundation of

407

China (grant no. 31270979 to X.R.), the new drug development project of China

408

(grant no. 2012ZX09103301-038 to X.R.) and the translational project of TMMU

409

(grant no. 2016XZH01 to X.R.). The funders had no role in study design, data

410

collection and analysis, decision to publish or preparation of the manuscript.

411 412 413

REFERENCES

414

(1) Leveque, N.; Semler, B. L. PLoS Pathog. 2015,11, e1004825.

415

(2) Terlizzi, M. E.; Occhipinti, A.; Luganini, A.; Maffei, M. E.; Gribaudo, G. Antiviral Res.

416 417 418 419 420

2016,132, 154–164. (3) Tummers, B.;Goedemans, R.; Pelascini, L. P.; Jordanova, E. S.;van Esch, E. M.; Meyers, C.; Melief, C. J.; Boer, J. M.; van der Burg, S. H. Nat. Commun. 2015, 6, 6537. (4) Lim, X. X.; Chandramohan, A.; Lim, X. Y.; Bag, N.; Sharma, K. K.; Wirawan, M.; Wohland, T.; Lok, S. M.; Anand, G. S. Nat. Commun. 2017, 8, 14339.

421

(5) Yauch, L. E.; Shresta, S. Adv. Virus Res. 2014, 88, 315–372.

422

(6) Bishop, D. G.; Work, E. Biochem. J. 1965, 96, 567–576. 18

ACS Paragon Plus Environment

Page 19 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

423 424 425 426

(7) Petersen, H.; Nieves, W.; Russell-Lodrigue, K.; Roy, C. J.; Morici, L. A. Procedia Vaccinol.

2014, 8, 38–42. (8) Mitra, S.; Barman, S.; Nag, D.; Sinha, R.; Saha, D. R.; Koley, H. FEMS Immunol. Med. Microbiol. 2012, 66, 240–250.

427

(9) Muralinath, M.; Kuehn, M. J.; Roland, K. L.; Curtiss, R. Infect. Immun. 2011, 79, 887–894.

428

(10) Bai, X.; Findlow, J.; Borrow, R. Expert. Opin. Biol. Ther. 2011, 11, 969–985.

429

(11) Lee, D. H.; Kim, S. H.; Kang, W.; Choi, Y. S.; Lee, S. H.; Lee, S. R.; You, S.; Lee, H.

430

K.; Chang, K. T.; Shin, E. C. Vaccine. 2011, 29, 8293–8301.

431

(12) Bartolini, E.;Ianni, E.; Frigimelica, E.; Petracca, R.; Galli, G.; Berlanda Scorza, F.; Norais,

432

N.; Laera, D.; Giusti, F.; Pierleoni, A.; Donati, M.; Cevenini, R.; Finco, O.; Grandi, G.;

433

Grifantini, R. J. Extracell. Vesicles. 2013, 2.

434

(13) Bonnington, K. E.; Kuehn, M. J. Biochim. Biophys. Acta. 2014, 1843, 1612–1619.

435

(14) Lee, E. Y.; Choi, D. Y.; Kim, D. K.; Kim, J. W.; Park, J. O.; Kim, S.; Kim, S.

436

H.; Desiderio, D. M.; Kim, Y. K.; Kim, K. P.; Gho, Y. S. Proteomics. 2009, 9, 5425–

437

5436.

438 439

(15) Brown, L.; Kessler, A.; Cabezas-Sanchez, P.; Luque-Garcia, J. L.; Casadevall, A. Mol. Microbiol. 2014, 93, 183–198.

440

(16) Beveridge, T. J. J. Bacteriol. 1999, 181, 4725–4733.

441

(17) Kim, J. H.; Lee, J.; Park, J.; Gho, Y. S. Semin. Cell Dev. Biol. 2015, 40, 97–104.

442

(18) Prados-Rosales, R.; Carreño,L. J.; Batista-Gonzalez, A.; Baena, A.; Venkataswamy, M.

443

M.; Xu, J.; Yu, X.; Wallstrom, G.; Magee, D. M.; LaBaer, J.; Achkar, J. M.; Jacobs, W. R.

444

Jr.; Chan, J.; Porcelli, S. A.; Casadevall, A. MBio. 2014, 5, e01921-14.

445 446

(19) Choi, C. W.; Park, E. C.; Yun, S. H.; Lee, S. Y.; Kim, S. I.; Kim, G. H. J. Immunol. Res. 2017, 2017, 7931982.

447

(20) Roier, S.; Zingl, F. G.; Cakar, F.; Schild, S. Microbial cell (Graz, Austria) 2016, 3, 257–259.

448

(21) Prados-Rosales, R.; Brown, L.; Casadevall, A.; Montalvo-Quirós, S.; Luque-Garcia, J. L.

449

Methods X. 2014, 1, 124–129.

450

(22) Traber, K.; Novick, R. Mol. Microbiol. 2006, 59, 1519–1530.

451

(23) Waldron, D. E.; Lindsay, J. A. J. Bacteriol. 2006, 188, 5578–5585.

452

(24) Gurung, M.; Moon,D. C.; Choi, C. W.; Lee, J. H.; Bae, Y. C.; Kim, J.; Lee, Y. C.; Seol, S. Y.; 19

ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

453

Cho, D. T.; Kim, S. L.; Lee, J. C. PloS One. 2011, 6, e27958.

454

(25) Thay, B.; Wai, S. N.; Oscarsson, J. PloS One. 2013, 8, e54661.

455

(26) Rivera, J.; Cordero, R. J.; Nakouzi, A. S.; Frases, S.; Nicola, A.; Casadevall, A. Proc. Natl.

456

Acad. Sci. U.S.A. 2010, 107, 19002–19007.

457

(27) Wang, B.; Muir, T. W. Cell Chem. Biol. 2016, 23, 214–224.

458

(28) Jeon, H.; Oh, M. H.; Jun, S. H.; Kim, S. I.; Choi, C. W.; Kwon, H. I.; Na, S. H.; Kim, Y. J.;

459

Nicholas, A.; Selas, G. N.; Lee, J. C. Microb. Pathog. 2016, 93, 185–193.

460

(29) Keiser, P. B.; Gibbs, B. T.; Coster, T. S.; Moran, E. E.; Stoddard, M. B.; Labrie 3rd, J.

461

E.; Schmiel, D. H.; Pinto, V.; Chen, P.; Zollinger; W. D. Vaccine 2010, 28, 6970–6976.

462

(30) Bhatt, S.; Gething, P. W.; Brady, O. J.; Messina, J. P.; Farlow, A. W.; Moyes, C. L.; Drake, J.

463

M.; Brownstein, J. S.; Hoen, A. G.; Sankoh, O.; Myers, M. F.; George, D. B.; Jaenisch, T.;

464

Wint, G. R.; Simmons, C. P.; Scott, T. W.; Farrar, J. J.; Hay, S. I. Nature. 2013, 496, 504-507.

465

(31) Crill, W. D.; Roehrig, J. T. J. Virol. 2001, 75, 7769–7773.

466

(32) Wahala, W. M.; Donaldson, E. F.; de Alwis, R.; Accavitti-Loper, M. A.; Baric, R. S.; de Silva,

467

A. M. PLoS Pathog. 2010, 6, e1000821.

468

(33) Bowen, D. M.; Lewis, J. A.; Lu, W.; Schein, C. H. Vaccine. 2012,30, 6081–6087.

469

(34) Danecek, P.; Lu, W.; Schein, C. H. J. Mol. Biol. 2010, 396, 550–563.

470

(35) Lok, S. M.; Kostyuchenko, V.; Nybakken, G. E.; Holdaway, H. A.; Battisti, A.

471

J.; Sukupolvi-Petty, S.; Sedlak,

D.; Fremont, D. H.; Chipman, P. R.; Roehrig, J. T.;

472

Diamond, M. S.; Kuhn, R. J.; Rossmann, M. G. Nat. Struct. Mol. Biol. 2008, 15, 312–317.

473 474 475 476 477 478 479 480 481 482 20

ACS Paragon Plus Environment

Page 20 of 21

Page 21 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

483 484

Table of Contents Figure

485

486 487 488 489 490 491 492 493 494 495

21

ACS Paragon Plus Environment